Antibacterial composition having xanthorrizol

ABSTRACT

An antibacterial composition having xanthorrizol is provided. The xanthorrizol is prepared by obtaining crude extract having antibacterial activity from  Curcuma xanthorriza  Roxb. lysate by the aid of solvent extraction, supercritical fluid extraction, microwave extraction or ultrasonic extraction; applying the crude extract to chromatography to obtain active fraction; acetylation of the active fraction to change its polarity and applying to chromatography to isolate an acetylated single compound; and, deacetylation of the acetylated single compound to give xanthorrizol having antibacterial activity. Since xanthorrizol has potential antibacterial activity over a broad spectrum of microorganisms under a wide range of temperature, it can be practically applied to antibacterial agent, tooth paste, oral cleanser, chewing gum, soap and cosmetics which require the antibacterial activity.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for preparing xanthorrizoland novel uses of the same, more specifically, to a process forpreparing xanthorrizol from Curcuma xanthorriza Roxb. and uses ofxanthorrizol as active ingredients for antibacterial agent, tooth paste,oral cleanser, chewing gum, soap and cosmetics.

2. Description of Related Art

Caries and periodontal disease are infectious dental diseases which areprimarily caused by dental pathogens such as Streptococcus mutans,Streptococcus sorbrinus, Actinomyces viscosus and Porphyromonasgingivalis(see: Marsh P. D., et al., J. Dent. Res., 71:1431-1438, 1992).To prevent and treat these caries and periodontal disease, antibioticssuch as spiramycin, vancomycin and chlorohexidine, and organic/inorganicfluoride have been conventionally used in the art. The conventionalantibiotics are, however, proven to be less satisfactory in a sense thatthey may cause untoward effects such as drug resistance, diarrhea andemesis. Moreover, sodium chloride, antiplasmin, allantoin derivatives,vitamins and amino acids, which are major active ingredients for oralcleanser, are not sufficient enough for removing pathogens on teethsurface. Particularly, it has been well known that sodium chloridepossesses preventive and curable effects on caries and periodontaldisease. However, it has also revealed a shortcoming that it notablyreduces foaming power of anionic surfactant whose main purpose is tofacilitate cleansing effect, and lacks full satisfaction in terms ofrefresh feeling due to its salty taste. Natually, many researchers havetried to deveop the alternative substances with no adverse effects, foreradication of pathogens causing caries and peridontal disease.

On the other hand, acne, athlete's foot, itchiness and eczema are causedby skin infectious pathogens such as Propionibacterium acnes, Candidaalbicans, Staphylococcus aureus and Staphylococcus epidermis(see: RamanA., et al., Lett. Appl. Microbiol., 21:242-245, 1995). To inhibit thesaid skin infectious pathogens, benzoyl peroxide, salicylic acid,benzalkonium chloride and antibiotics such as erythromycin, and naturalproducts such as tea tree oil, royal jelly extract and ginseng extracthave been used in the art. While the antibiotics may have some positiveeffects on the treatment of skin infections, they may cause drugresistance and other adverse effects such as rash and inflammation,which naturally limits their practical use(see: Gollnick H., et al., J.Dermatology, 196:119-125, 1998). Moreover, upon therapeutic applicationfor dermal diseases or cosmetics, antibiotics derived from naturalresources may cause unexpected decline in their efficacy, since they arenot single compounds. Also, application of these natural products has acertain limitation since they have a narrow spectrum of antibacterialactivity, and their activities may be decreased by evaporation ordegradation at high temperature caused by weak thermal stability(see:Higaki S., et al., J. Dermatology, 23:310-314, 1996). Under thecircumstances, there are strong reasons for exploring and developingsubstances having high antibacterial activity on skin infections with noadverse effects from natural products such as medicinal plants.

SUMMARY OF THE INVENTION

The present inventors have made efforts to develop a substance fromnatural source which can efficiently inhibit the dental infections andskin diseases, and finally discovered that: xanthorrizol prepared fromCurcuma xanthorriza Roxb. has a wide spectrum of antibacterial activity,heat stability and safety onto human skin; and, therefore, it can beapplied in antibacterial agent, tooth paste, oral cleanser, soap andcosmetics which require antibacterial activity.

The present invention provides a process for preparing xanthorrizol fromCurcuma xanthorriza Roxb. and an antibacterial composition havingxanthorrizol.

The invention provides a process for using xanthorrizol as an activeingredient of antibacterial agent.

The invention provides a process for using xanthorrizol as an activeingredient of tooth paste.

The invention provides a process for using xanthorrizol as an activeingredient of oral cleanser.

The invention provides a process for using xanthorrizol as an activeingredient of chewing gum.

The invention provides a process for using xanthorrizol as an activeingredient of antibacterial soap.

The invention provides a process for using xanthorrizol as an activeingredient of cosmetics.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects, and features of the present inventionwill become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

FIG. 1 is a graph showing antibacterial activity of crude extract fromCurcuma xanthorriza Roxb. against Streptococcus mutans in terms ofviable cell count.

FIG. 2 is a graph showing antibacterial activity of purifiedxanthorrizol against Propionibacterium acnes in terms of viable cellcount.

FIG. 3 is a graph showing antibacterial activity of purifiedxanthorrizol against Streptococcus mutans using viable cell count.

FIG. 4a is a graph showing the absorbance change by the efflux ofnucleic acid from Streptococcus mutans after xanthorrizol treatment.

FIG. 4b is a graph showing the absorbance change by the efflux ofprotein from Streptococcus mutans after xanthorrizol treatment.

FIG. 5a is a graph showing the absorbance change by the efflux ofcalcium from Streptococcus mutans after xanthorrizol treatment.

FIG. 5b is a graph showing absorbance change by the efflux of magnesiumfrom Streptococcus mutans after xanthorrizol treatment.

DETAILED DESCRIPTION OF THE INVENTION

The process for preparing xanthorrizol of the present inventioncomprises the steps of: obtaining crude extract having antibacterialactivity from Curcuma xanthorrhiza Roxb. by the aid of solventextraction, supercritical fluid extraction, microwave extraction, orultrasonication; applying the crude extract thus isolated to a columnchromatography to obtain active fraction; acetylating the activefraction and applying the said fraction to a column chromatography toisolate an acetylated single compound; and, deacetylating the acetylatedsingle compound to give xanthorrizol having antibacterial activity.

The process for preparing xanthorrizol of present invention comprisesthe following steps:

Step 1: Isolation of Crude Extract

Lysate of Curcuma xanthorriza Roxb. is extracted by one of extractionmethods employing organic solvent, supercritical fluid, microwave orultrasonic wave, and subsequently extracted again with ethylacetate toisolate crude extract having antimicrobial activity. The solventextraction is carried out by employing an organic solvent of methanol,ethanol, hexane, ethylacetate or chloroform; water; or, mixturesthereof, preferably, water or 50 to 100%(v/v) methanol, ethanol orhexane, most preferably, 50 to 100%(v/v) methanol. The supercriticalfluid extraction is performed by employing a supercritical fluid ofcarbon dioxide at 35 to 70° C. under a pressure of 100 to 400 bar; themicrowave extraction, by employing a solvent of water, methanol, ethanolor hexane under a microwave condition of 2,450 MHz for 1 to 10 minutesat 50 to 120° C.; and, the ultrasonic extraction, by employing a solventof water, methanol, ethanol or hexane under an ultrasonic condition of48 kHz for 10 to 60 minutes at 15 to 50° C. The crude extract isfiltrated using Whatman filtering paper, condensed using rotaryevaporator under a reduced pressure to remove the solvent, furtherextracted with ethylacetate, and condensed again using rotary evaporatorunder a reduced pressure to give crude extract having antibacterialactivity.

Step 2: Silica Gel Chromatography(I)

The crude extract prepared in the previous step is applied to a columnchromatography to obtain active fraction with antibacterial activity:silica gel is employed as a resin for the chromatography and elution ismade by a solvent mixture of hexane and ethylacetate, hexane andchloroform, or hexane and benzene, preferably, a mixture of hexane andethylacetate(1:1 to 50:1(v/v)), most preferably, a mixture of hexane andethylacetate(1:1 to 10:1(v/v)), and optionally stepwise elution bychanging a mixing ratio of solvents with the lapse of time.

Step 3: Acetylation/Chromatography(II)

The active fraction obtained in the previous step is acetylated andapplied to a column chromatography again to isolate a single compoundhaving antibacterial activity. The acetylation is carried out bydissolving the fraction in pyridine, adding the equal volume of aceticanhydride, and reacting for more than 15 hours, preferably, 15 to 36hours at room temperature. Then, the silica gel chromatography iscarried out to isolate an acetylated single compound with antibacterialactivity by employing a mixture of hexane and ethylacetate, hexane andchloroform, hexane and benzene, or mixtures of other organic solvents,preferably, a mixture of hexane and ethylacetate(5:1 to 50:1(v/v)).

Step 4: Deacetylation for Obtaining Xanthorrizol

The acetylated single compound isolated in the previous step isdissolved in methanol, deacetylated by the addition of KOH, andneutralized by applying it onto a cation-exchange resin to givexanthorrizol having antibacterial activity. Preferably, concentration ofKOH is controlled at a range of 3 to 7%, and the deacetylation isperformed for more than 2 hours, preferably 2 to 4 hours, mostpreferably 3 hours.

By analyzing functional groups with ¹H-NMR, ¹³C-NMR(4000 MHz, CDCl₃) IR,and measuring molecular weight with EIMS(electronic isolation massspectrometry), the purified single compound was finally identified asxanthorrizol, a kind of sequiterpenoids having biasabolane structurerepresented by the following formula.

Based on the measurement of MIC value, xanthorrizol of the presentinvention was proven to show strong antibacterial activity over a broadspectrum of microorganisms. Well diffusion assay also revealed that itis highly stable at a high temperature. In addition, xanthorrizol wasfound to have safety through patch test onto human skin. Therefore,xanthorrizol of the present invention, with its strong antibacterialactivity and safety, may be formulated into antibacterial agent, toothpaste, oral cleanser, chewing gum, soap and cosmetics which requireantibacterial activity.

To prepare the antibacterial agent of the present invention,xanthorrizol may be formulated into tablet, capsule, soft gelatincapsule, solution, ointment, plaster, granule, lotion, aerosol,suppository, cataplasma, cream, troche, paste, pill and injection bymixing it with pharmaceutically acceptable carriers such aspolyvinylpyrrolidone and hydroxypropylcellulose; disintegrators such ascalcium carboxymethylcellulose and sodium glycolate starch; diluentssuch as corn starch, lactose, soy bean oil, cellulose crystal andmannitol; lubricants such as magnesium stearic acid and talc; sweetenerssuch as sugar, fructose, sorbitol and aspartame; stabilizers such assodium carboxymethylcellulose, α or β cyclodextrin, vitamin C, citricacid, white wax; preservatives such as methyl p-oxybenzoic acid, propylp-oxybenzoic acid and sodium benzoic acid; and spice such as ethylvaseline, masking flavor, menthol and herb. These antibacterial agentsmay serve as effective therapy for dental caries, periodontal disease,acne, athlete's foot, itchiness and eczema.

Xanthorrizol having antibacterial activity may be applied in thepreparation of tooth paste, oral cleanser and chewing gum. For example,in case of oral cleanser, it may comprise xanthorrizol as an activeingredient in a concetration of 0.0001 to 0.1%(w/w) together with theconventional ingredients for oral cleansers such as abrasive, binder,foamer, spice, sweetner, buffer and alcohol. Similarly, tooth paste canbe formulated comprising xanthorrizol in a concentration of 0.001 to1.0%(w/w) with the ingredients such as abrasive, fluoride compound as anadditional active ingredient, binder, foamer, spice, sweetener andbuffer. For the preparation of chewing gum, it may further compriseingredients such as gum base, sugar powder, glucose, starch hydrolysate,glycerin and spice.

Moreover, antibacterial soap containing xanthorrizol may be manufacturedby employing conventional soap base which include fat, coconut oil, palmoil and glycerin, together with spice, pigments, antioxidants, andmoisteners. The content of xanthorrizol in the antibacterial soap ispreferred in a range of 0.001 to 5.0%(w/w) for the following reasons:less than 0.001% of xanthorrizol may decrease antibacterial anddisinfectant activity which in turn reduces its efficiency on eczema,itchiness, acne and freckle, while more than 5.0% may reduce thesynergic effects on the whole and decrease the cleansing effects due torelative low contents of soap base.

Meanwhile, cosmetics such as facial lotion and cream can be manufacturedby containing xanthorrizol in a concentration of 0.001 to 1.0%(w/w),wherein the lotion may further comprise the conventional ingredients oflotion such as abrasive, buffer, distilled water, glycerin, spice, oiland alcohol. In case of the facial cream, xanthorrizol may be includedin a concentration of 0.001 to 3.0%(w/w) together with the conventionalingredients for facial cream such as abrasive, distilled water,glycerin, buffer, spice and oil.

The present invention is further illustrated in the following examples,which should not be taken to limit the scope of the invention.

EXAMPLE 1 Preparation of Crude Extract Example 1-1

Preparation of Crude Extract Having Antibacterial Activity

100 g of Curcuma xanthorriza Roxb. lysate was repeatedly extracted with400 ml of 75%(v/v) methanol at room temperature for two days. Then, theextract was filtered through Whatman filter paper(No. 2), condensedusing rotary evaporator(Heidolph VV2011, Switzerland) to eliminatesolvent, and freeze-dried to obtain 11.5 g of methnol extract.

The methanol extract was fractionated based on the polarity of organicsolvents as following: the methanol extract was condensed to removesolvent, extracted with 4 volumes of ethylacetate twice to give 4.8 g ofethylacetate fraction. Then, 1.7 g of butanol extract was obtained bythe same procedure employing butanol. Subsquently, 1.1 g of waterfraction was yielded. To measure the antibacterial activity, each of themethanol fraction, ethylacetate fraction, butanol fraction and waterfraction was dissolved in dimethylsulfoxide(DMSO) to give specimens in aconcentration of 2%, 1% and 0.1% for each fraction.

To measure the antibacterial activity, Streptococcus mutans(ATCC 25175,U.S.A.) was stored on BHI(brain heart infusion, Difco Co., U.S.A.)medium containing 1.5% agar at 4° C. and subcultured at one monthinterval. Antibacterial activity of the specimens prepared above wasmeasured by well diffusion assay: 5 ml of top agar medium includingStreptococcus mutans was poured onto the solid medium preparedpreviously. After the top agar medium was hardened, wells were made byboring the top agar medium on its surface with a sterilized tip. Then,0.1 ml of samples in 10% DMSO were loaded into the wells and allowed todiffuse for more than 2 hours. The solid medium in which samples areloaded were then incubated at 37° C. for 16 hours, and the antibacterialactivity of each sample was compared on the basis of diameter(mm) of theclear zone surrounding the well. DMSO was used as control group, and theresults are shown in Table 1.

TABLE 1 Antibacterial activity of solvent extracts Diameter of ClearZone (mm) Methanol Ethylacetate Butanol Water Conc. (%) Extract FractionFraction Fraction 2 18 20 — — 1 16 19 — — 0.1 15 15 — —

As shown in Table 1, ethylacetate fraction exhibited the highestantibacterial activity. Therefore, further purification of a singlecompound from the ethylacetate fraction was performed.

After eliminating solvent from the ethylacetate fraction with rotaryevaporator, the remaining substances were dissolved in 10% DMSO andassayed for antibacterial activity. The specimens were prepared in aconcentration of 0.001, 0.003, and 0.005%, treated with Streptococcusmutans at 37° C. for 1, 2, 5 and 10 minutes, respectively, and subjectedto “pour plate method” by serial 10-fold dilution to count viable cellnumbers of the bacteria. The viable cell numbers were compared to thoseof green tea extract which is well known for its preventive effect oncaries. FIG. 1 is a graph showing antibacterial activity of crudeextract from Curcuma xanthorriza Roxb. against Streptococcus mutans interms of viable cell numbers, where ♦ represents DMSO, control; ▪,0.001% crude extract; ▾, 0.003% crude extract; x, 0.005% crude extract,respectively, As shown in FIG. 1, almost all bacteria were killed withinone minute when the concentration of crude extract was 0.005%(50 μg/ml).Compared to conventional medicinal herb extracts whose minimuminhibitory concentrations are within the range of 200 to 1000 μg/ml theethylacetate crude extract exhibited higher antibacterial activity.

Example 1-2

Preparation of Tooth Paste Containing Crude Extract of Curcumaxanthorriza Roxb.

Tooth paste was prepared to contain the crude extract obtained inExample 1-1: alkyl sodium sulfate, sucrose fatty acid ester, sodiumcarboxymethylcellulose and sodium chloride were dispersed in glycerinhomogeneously. Then, distilled water was added to dilute the mixture,followed by the addition of sodium. The crude extract was added to themixture in a final concentration of 5.0, 1.0, 0.1 and 0.01%,respectively, and spice was added under a vacuum condition to givecream-type tooth pastes, whose particular ingredients are disclosed inTable 2.

TABLE 2 Tooth pastes containing crude extract of Curcuma xanthorrhizaRoxb. Tooth Tooth Tooth Tooth Ingredient Paste I Paste II Paste IIIPaste IV Crude Extract 5.0% 1.0% 0.1% 0.01% Sorbitol Solution 19.2%19.2% 19.2% 19.2% Glycerin 22.0% 22.0% 22.0% 22.0% Sucrose Fatty AcidEster 2.0% 2.0% 2.0% 2.0% Alkyl Sodium Sulfate 1.7% 1.7% 1.7% 1.7%Sodium Chloride 1.0% 1.0% 1.0% 1.0% Spice 1.1% 1.1% 1.1% 1.1% Sodium0.4% 0.4% 0.4% 0.4% Carboxymethylcellulose Distilled Water q.s. q.s.q.s. q.s.

Each of the tooth paste containing the crude extract was taken totest-tubes, dissolved in 4 ml of medium for dental pathogens, and mixedwith 1 ml of dental pathogen cultures. The test-tubes were incubatedwith shaking, and antibacterial activity was measured by counting viablecells with the lapse of time. Similarly, the antibacterial activity ofcrude extract-free tooth paste as a control was also determined againstthe dental pathogens. The results are shown in Table 3.

TABLE 3 Antibacterial activity of tooth pastes containing crude extractof Curcuma xanthorrhiza Roxb. Viable Cell Count (cell number/ml) DentalPathogen Test Solution 0 hr 24 hr Streptococcus Tooth Paste I 2.0 × 10⁵<1 mutans Tooth Paste II 2.0 × 10⁵ <1 Tooth Paste III 2.0 × 10⁵ <1 ToothPaste IV 2.0 × 10⁵ <1 Control 2.0 × 10⁵ 3.1 × 10⁴ Streptococcus ToothPaste I 9.1 × 10⁵ <1 sobrinus Tooth Paste II 9.1 × 10⁵ <1 Tooth PasteIII 9.1 × 10⁵ <1 Tooth Paste IV 9.1 × 10⁵ <1 Control 9.1 × 10⁵ 3.1 × 10⁴Porphyromonas Tooth Paste I 5.5 × 10⁵ <1 gingivalis Tooth Paste II 5.5 ×10⁵ <10  Tooth Paste III 5.5 × 10⁵ <15  Tooth Paste IV 5.5 × 10⁵ 6.7 ×10³ Control 5.5 × 10⁵ 3.1 × 10⁴ Actinomyces Tooth Paste I 8.2 × 10⁵ <1viscosus Tooth Paste II 8.2 × 10⁵ <1 Tooth Paste III 8.2 × 10⁵ <1 ToothPaste IV 8.2 × 10⁵ <15  Control 8.2 × 10⁵ 2.5 × 10⁴

As shown in Table 3, the tooth pastes containing the crude extractexhibited higher antibacterial activity against the dental pathogens,compared to that of control which does not contain the crude extract.

Example 1-3

Preparation of Oral Cleansers Containing Crude Extract of Curcumaxanthorrhiza Roxb.

To prepare oral cleanser, the crude extract was first mixed with waterin a concentration of 1.0, 0.1, 0.01 and 0.001%, respectively, andsubsequently with citrate solution and 1-menthol dissolved in ethanol.Then, sodium saccharin and sodium fluoride were dissolved in water, andadded to the said mixtures together with pigments, peppermint essenceand spearmint essence. And then, to the mixtures was added water to givehomogeneous mixtures with equal amounts, whose particular ingredientsare disclosed in Table 4.

TABLE 4 Oral cleansers containing crude extract of Curcuma xanthorrhizaRoxb. Oral Oral Oral Oral Oral Cleanser Cleanser I Cleanser II CleanserIII Cleanser IV Crude Extract 1.0% 0.1% 0.01%  0.001% Sodium Fluoride0.02%  0.02%  0.02%   0.02% Ethanol 6.6% 6.6% 6.6%  6.6% Glycerin 6.0%6.0% 6.0%  6.0% /-Menthol 0.005%  0.005%  0.005%  0.005% SodiumSaccharin q.s. q.s. q.s. q.s. Citric Acid q.s. q.s. q.s. q.s. Pigmentq.s. q.s. q.s. q.s. Peppermint q.s. q.s. q.s. q.s. Essence Spearmintq.s. q.s. q.s. q.s. Essence

4 ml of each oral cleanser containing the crude extract with differentconcentrations was taken to test-tubes, and mixed with 1 ml of dentalpathogen cultures. The test-tubes were incubated with shaking, andantibacterial activity was measured after 24 hours by counting viablecell numbers, whose results are shown in Table 5.

TABLE 5 Antibacterial activity of oral cleansers containing crudeextract of Curcuma xanthorrhiza Roxb. Viable Cell Count (cell number/ml)Dental Pathogen Test Solution 0 hr 24 hr Streptococcus Oral Cleanser I2.0 × 10⁵ <1 mutans Oral Cleanser II 2.0 × 10⁵ <1 Oral Cleanser III 2.0× 10⁵ <1 Oral Cleanser IV 2.0 × 10⁵ 1.1 × 10³ Control 2.0 × 10⁵ 2.5 ×10⁴ Streptococcus Oral Cleanser I 9.1 × 10⁵ <1 sobrinus Oral Cleanser II9.1 × 10⁵ <1 Oral Cleanser III 9.1 × 10⁵ <1 Oral Cleanser IV 9.1 × 10⁵2.5 × 10² Control 9.1 × 10⁵ 2.5 × 10⁴ Porphyromonas Oral Cleanser I 5.5× 10⁵ <5 gingivalis Oral Cleanser II 5.5 × 10⁵ <15  Oral Cleanser III5.5 × 10⁵ 1.1 × 10² Oral Cleanser IV 5.5 × 10⁵ 1.2 × 10³ Control 5.5 ×10⁵ 2.5 × 10⁴ Actinomyces Oral Cleanser I 8.2 × 10⁵ <1 viscosus OralCleanser II 8.2 × 10⁵ <1 Oral Cleanser III 8.2 × 10⁵ <10  Oral CleanserIV 8.2 × 10⁵ 4.5 × 10³ control 8.2 × 10⁵ 2.5 × 10⁴

As clearly demonstrated in Table 5, oral cleansers containing crudeextract of Curcuma xanthorrhiza Roxb. were proven to have higherantibacterial activity against the dental pathogens causing caries,compared to that of control groups.

Example 1-4

Preparation of Chewing Gum Containing Crude Extract of Curcumaxanthorrhiza Roxb.

Gum base, starch hydrolysate, glucose, glycerin and sugar powder weremixed in a prewarmed blender. Then, to the mixture was added the crudeextract in a final concentration of 1.0, 0.1, 0.01 and 0.001%,respectively, and mixed with spice and distilled water in the blenderwhose inner temperature was maintained at 55° C. for 20 minutes, finallyto give chewing gum. Particular ingredients of the chewing gum thusprepared are disclosed in Table 6.

TABLE 6 Chewing gum containing crude extract of Curcuma xanthorrhizaRoxb. Chewing Chewing Chewing Chewing Chewing Gum Gum I Gum II Gum IIIGum IV Crude Extract 1.0%  0.1%  0.01%   0.001%   Gum Base 20% 20% 20%20% Sugar Powder 54% 54% 54% 54% Starch 10% 10% 10% 10% HydrolysateGlycerin  5%  5%  5%  5% Spice q.s. q.s. q.s. q.s. Distilled Water q.s.q.s. q.s. q.s.

Example 1-5

Preparation of Facial Lotion Containing Crude Extract of Curcumaxanthorrhiza Roxb.

To prepare facial lotion containing crude extract of Curcumaxanthorrhiza Roxb., the crude extract was first mixed with water in 4different concentrations of 5.0, 1.0, 0.1 and 0.01%, respectively, andsubsequently with phosphate solution. Then, ethanol, glycerin andpropyleneglycol were added to the said mixtures together with spices andpreservatives. And then, to the mixtures was added water to givehomogeneous mixtures with equal amounts, whose particular ingredientsare disclosed in Table 7.

TABLE 7 Facial lotion containing crude extract of Curcuma xanthorrhizaRoxb. Facial Lotion Lotion I Lotion II Lotion III Lotion IV Crudeextract 5.0% 1.0% 0.1% 0.01%  Glycerin 2.0% 2.0% 2.0% 2.0%Propyleneglycol 2.0% 2.0% 2.0% 2.0% Potassium phosphate 0.1% 0.1% 0.1%0.1% Disodium phosphate 0.05%  0.05%  0.05%  0.05%  Spice 0.02%  0.02% 0.02%  0.02%  96% ethanol  20%  20%  20%  20% Distilled water q.s. q.s.q.s. q.s. Preservative q.s. q.s. q.s. q.s.

4 ml of each facial lotion containing the crude extract was taken totest-tubes, and mixed with 1 ml of acne-causing pathogen cultures. Thetest-tubes were incubated with shaking, and antibacterial activity wasmeasured after 24 hours by counting viable cell numbers, whose resultsare shown in Table 8.

TABLE 8 Antibacterial activity of facial lotion containing crude extractof Curcuma xanthorrhiza Roxb. Acne-causing Viable Cell Count (cellnumber/ml) Pathogen Test Solution 0 hr 24 hr Propionibacterium Lotion I3.0 × 10⁵ <1 acnes Lotion II 3.0 × 10⁵ <1 Lotion III 3.0 × 10⁵ <1 LotionIV 3.0 × 10⁵ 1.1 × 10² Control 3.0 × 10⁵ 3.1 × 10³ Staphylococcus LotionI 4.5 × 10⁵ <1 aureus Lotion II 4.5 × 10⁵ <1 Lotion III 4.5 × 10⁵ <1Lotion IV 4.5 × 10⁵ 4.1 × 10² Control 4.5 × 10⁵ 4.7 × 10³ StaphlococcusLotion I 6.8 × 10⁵ <1 epidermis Lotion II 6.8 × 10⁵ <1 Lotion III 6.8 ×10⁵ <1 Lotion IV 6.8 × 10⁵ 4.4 × 10² Control 6.8 × 10⁵ 5.1 × 10³

As clearly demonstrated in Table 3, facial lotion containing crudeextract of Curcuma xanthorrhiza Roxb. was proven to have higherantibacterial activity, compared to that of control groups.

Example 1-6

Preparation of Facial Cream Containing Crude Extract of Curcumaxanthorrhiza Roxb.

Facial creams containing the crude extract were prepared in accordancewith the components and composition ratios shown in Table 9 below:first, substances B and C were each melted down at the temperature of 75to 80° C., followed by emulsification of the melted substance C in themelted substance B. Then, xanthorrizol was added to the mixtures in aconcentration of 10.0, 1.0, 0.1 and 0.01%, respectively. Finally, spicewas added to the mixtures which were adjusted to the final volume withdistilled water.

TABLE 9 Facial creams containing crude extract of Curcuma xanthorrhizaRoxb. Facial Facial Facial Facial Facial Cream Cream I Cream II CreamIII Cream IV A Crude Extract 10.0%  1.0% 0.1% 0.01%  Glycerin 2.0% 2.0%2.0% 2.0% Propyleneglycol 2.0% 2.0% 2.0% 2.0% B Chlorolauryl 8.0% 8.0%8.0% 8.0% Sulfate Stearin 5.4% 5.4% 5.4% 5.4% Mineral Oil 4.5% 4.5% 4.5%4.5% C Spice 0.02%  0.02%  0.02%  0.02%  Cetyl Alcohol 6.5% 6.5% 6.5%6.5% Distilled Water q.s. q.s. q.s. q.s. Preservative q.s. q.s. q.s.q.s.

4 ml of each cream containing the crude extract was taken to test-tubes,and mixed with 1 ml of acne-causing pathogen cultures. The test-tubeswere incubated with shaking, and antibacterial activity was measuredafter 24 hours by counting viable cell numbers, whose results are shownin Table 10.

TABLE 10 Antibacterial activity of facial creams containing crudeextract of Curcuma xanthorrhiza Roxb. Viable Cell Count Acne-causing(cell number/ml) Pathogen Test Solution 0 hr 24 hr PropionibacteriumFacial Cream I 3.0 × 10⁵ <1 acnes Facial Cream II 3.0 × 10⁵ <1 FacialCream III 3.0 × 10⁵ <1 Facial Cream IV 3.0 × 10⁵ 1.3 × 10² Control 3.0 ×10⁵ 3.3 × 10³ Staphylococcus Facial Cream I 4.5 × 10⁵ <1 aureus FacialCream II 4.5 × 10⁵ <1 Facial Cream III 4.5 × 10⁵ <1 Facial Cream IV 4.5× 10⁵ 3.7 × 10⁴ Control 4.5 × 10⁵ 4.1 × 10³ Staphlococcus Facial Cream I6.8 × 10⁵ <1 epidermis Facial Cream II 6.8 × 10⁵ <1 Facial Cream III 6.8× 10⁵ <1 Facial Cream IV 6.8 × 10⁵ 4.3 × 10² Control 6.8 × 10⁵ 5.1 × 10³

As clearly demonstrated in Table 10, facial creams containing crudeextract of Curcuma xanthorrhiza Roxb. were proven to have higherantibacterial activity against acne-causing pathogens, compared to thatof control groups.

EXAMPLE 2 Isolation and Purification of Xanthorrizol HavingAntibacterial Activity

The crude extract, prepared in Example 1-1 was applied to a silica gelcolumn chromatography: the crude extract was applied to a columnchromatography(5×43 cm) filled with silica gel(Merck, U.S.A.) of 70 to230 mesh, and eluted with a mixed gradient of hexane and ethylacetate(10:1→3:1→1:1). Then, thin layer chromatography(TLC) was performed withthe active fractions obtained in the course of chromatographic procedureto give 8 different fractions(fractions I to VIII ) with similarretention on the TLC plate. Antibacterial activity of the fractions wasmeasured in terms of minimum inhibitory concentration(MIC), and comparedto that of vancomycin. The results are shown in Table 11.

TABLE 11 Minimum inhibitory concentration of active fractions FractionMIC (μg/ml) Fraction I 250 Fraction II  7 Fraction III  62 Fraction IV125 Fraction V 250 Fraction VI 250 Fraction VII No inhibitory actionFraction VIII No inhibitory action Vancomycin  1

Fraction II which possesses the strongest antibacterial activity waspooled to isolate substances having antibacterial activity. Sincefraction II had some impurities, acetylation of the fraction was carriedout to change its polarity as a procedure to obtain a single compoundwith antibacterial activity: 0.5 g of fraction II was dissolved in 5 mlpyridine, added with 5 ml of acetic anhydride in a drop-wise manner atice-cold temperature, and stirred at room temperature for more than 15hours. The reactant was subjected to partition extraction with 100 ml ofice-cold water once, and subsequently 100 ml of ethylacetate twice. Theethylacetate phase thus obtained was washed with 5% HCl, NaHCO₃ andNaCl, dehydrated with anhydrous MgSO₄, filtered and condensed under areduced pressure. The resultant was finally applied onto a silica gelcolumn(4.7×50 cm), and eluted with a solvent mixture of hexane andethylacetate(15:1, v/v) to give an acetylated single compound. Since thesingle compound showed no antibacterial activity in its acetylated form,deacetylation was performed by dissolving the acetylated single compoundin 20 ml of methanol. Then, 2 ml of 5% KOH was added to the compound,reacted with stirring for more than 2 hours, and neutralized usingcation exchange resin(DOWEX50WX4-400), followed by filtration andcondensation to give a single isolated compound. Thereafter, itsmolecular weight was measured with EI-MS(VG Platform II, FISIONS, 15eV), and chemical identification was conducted using analyticalinstruments of ¹H-NMR, ¹³C-NMR(400 MHz, CDCl₃) and IR(Perkin Elmerspectrum I , DTGS detector). The results of the analyses were asfollows: IR (CDCl₃, v, max) 3402, 2915, 1708, 1620, 1599 cm⁻¹;EI-MS(m/z) 218, 148, 136, 135, 121; ¹H-NMR(CDCl₃, 400 MHz): 1.18(3H, d,J=7.1 Hz, H-15), 1.52(3H, s, H-13), 1.57(2H, dt, J=7.1, 7.2 Hz, H-8),1.67(3H, s, H-12), 1.85(2H, dt, J=7.0, 7.2 Hz, H-9), 2.20(3H, s, H-14),2.59(1H, qt, J=7.1, 7.1 Hz, H-7), 5.08(1H, t, J=7.0 Hz, H-10), 6.59(1H,br s, H-2), 6.66(1H, br d, J=7.6 Hz, H-6), 7.01(1H, d, J=7.6 Hz, H-5);¹³C-NMR(CDCl₃, 400 MHz): 147.16(s, C-1), 113.50(d, C-2), 153.51(s, C-3),120.86(s, C-4), 130.74(d, C-5), 119.42(d, C-6), 38.98(d, C-7), 38.32(t,C-8), 26.10(t, C-9), 124.48(d, C-10), 131.39(s, C-11), 15.31(q, C-12),25.67(q, C-13), 17.64(q, C-14), 22.34(q, C-15). The ¹H-NMR spectrumshowed 2- or 3-substituted hydroxy-α-curcumene from its aromatic protonsignal. Also, through the analysis of chemical shift of ¹³C-NMR signal,the compound was found to have 1,3,4-substituted benzene ring system.Taken together, the compound was identified asxanthorrizol(1,3,5,10-bisabolatetraen-3-ol), a sequiterpenoid havingbisabolane structure, which is colorless oily material having theoptical rotation of [α]=−50.2°(C=0.65, CHCl₃).

EXAMPLE 3 Heat Stability and Antibacterial Activity of XanthorrizolExample 3-1

Antibacterial Activity of Xanthorrizol

To measure the antibacterial activity of xanthorrizol, it was dissolvedin DMSO to give specimens with a concentration of 0.001%, 0.0015% and0.002%, respectively, and incubated with test microorganisms(2×10CFU/ml) at 37° C. for 1, 2, 5, and 10 hours, respectively. Then, theywere subjected to serial 10-fold dilution, incubated with anacne-causing pathogen, Propionibacterium acnes for 24 to 48 hours, andcounted the viable cell number of the pathogen. FIG. 2 is a graphshowing antibacterial activity of xanthorrizol in terms of viable cellnumbers, where ♦ represents DMSO, control; ▪, 0.001% crude extract; ▾,0.0015% crude extract; x, 0.002% crude extract, respectively. As clearlydemonstrated in FIG. 2, the microorganism was eradicated almostcompletely in one hour at the concentration of xanthorrizol of 0.002%,indicating that xanthorrizol exerts strong antibacterial activityagainst Propionibacterium acnes at low concentrations.

Xanthorrizol was also tested for Streptococcus mutans in a similarmanner as described above: xanthorrizol was dissolved in DMSO to givespecimens with a concentration of 0.0002%, 0.0004% and 0.0008%,respectively. Then, the specimens were incubated with the microorganismat 37° C. for 1, 2, 5 and 10 minutes, respectively, and subjected toserial 10-fold dilution. After the incubation with the pathogen, viablecell number for Streptococcus mutans was counted. FIG. 3 is a graphshowing antibacterial activity of xanthorrizol against Streptococcusmutans in terms of viable cell number, where ♦ represents DMSO, control;▪, 0.0002% crude extract; Δ, 0.0004% crude extract; x, 0.0008% crudeextract, respectively. As shown in FIG. 3, the microorganism waseradicated almost completely in one minute at the concentration ofxanthorrizol of 0.0004%(4 μg/ml), demonstrating that xanthorrizolexhibits strong antibacterial activity against Streptococcus mutans atlow concentrations.

In addition, minimum inhibitory concentration(MIC, μg/ml) for variousmicroorganisms was determined by performing 2-fold dilution: first, 1 mlof medium was taken into each tube, and 1 ml of xanthorrizol(0.05% inDMSO) was added to only one tube to make 2-fold dilution. Then, serial2-fold dilution was further carried out for the rest of the tubes,starting from the tube containing xanthorrizol. 100 μl of microorganismwas added into each tube prepared by the 2-fold dilution to contain afinal cell number of 2×10⁵ CFU/ml, and incubated at 30 to 37° C. formore than 24 hours to determine minimum inhibitory concentrations forvarious test microorganisms(see: Table 12).

TABLE 12 Minimum inhibitory concentration (MIC, μg/ml) of xanthorrizolover various test microorganisms Microorganism MIC Microorganism MICActinomyces viscosus 16 Pityrosporum 125 ATCC 15988 pacchydermatis ATCC14522 Bifidobacterium bifidum 250 Porphyromonas gingivalis 32 ATCC 29521W 50 Candida albicans 125 Propionibacterium acnes 16 ATCC 10231 ATCC6919 Candida glabrata 125 Saccharomyces cerevisiae 250 IFO 0622 ATCC9763 Cladosporium cladosporioides 500 Salmonella typhimurium 62.5 IFO6348 IFO 12529 Enterococcus faecalis 16 Staphylococcus aureus 62.5 ATCC19433 ATCC 12600 Lactobacillus casei 250 Streptococcus mutans 2 ATCC4646 ATCC 25175 Lactobacillus aciophilus 500 Streptococcus sobrinus 4ATCC 4356 ATCC 27351 Penicilium chrysogenum 125 Streptococcus salivarius4 IFO 5472 ATCC 9758 Penicilium citrinum 250 Streptococcus sanguis 4 IFO6352 ATCC 35105 Streptomyces bikiniensis 32 ATCC 11062

As shown in Table 12, it was clearly demonstrated that xanthorrizolshows a broad spectrum of antibacterial activity.

Example 3-2

Thermal Stability of Xanthorrizol

Xanthorrizol was dissolved in DMSO to prepare specimens with aconcentration of 0.1%. The specimens were heated for 30 minutes at thetemperature of 60° C., 70° C., 80° C., 90° C., 100° C. and 121° C.,respectively. Then, antibacterial activity against Streptococcus mutanswas determined for each specimen by well diffusion analysis, whoseresults are shown in Table 13.

TABLE 13 Antibacterial activity of xanthorrizol at various temperaturesTreatment Temp. (° C.) 60 70 80 90 100 121 Diameter of Clear Zone (mm)17 16 17 17  18  17

As shown in Table 13, it was clearly demonstrated that xanthorrizol hasa strong antibacterial activity even under a high temperature of 60 to121° C.

Example 3-3

Efflux of Intracellular Macromolecules and Ions After XanthorrizolTreatment

Streptococcus mutans was treated with 5 and 10 μg/ml of xanthorrizol for1, 2, 5 and 10 minutes, respectively, to examine the efflux ofintracellular macromolecules and ions by the xanthorrizol-mediated celldamage. FIGS. 4a and 4 b are graphs showing the efflux of neucleic acidand protein from Streptococcus mutans after xanthorrizol treatment, andFIGS. 5a and 5 b, calcium efflux and magnesium efflux, where ♦represents 0.1% DMSO, control; ▪, Streptococcus mutans treated with 5μg/ml xanthorrizol; •, Streptococcus mutans treated with 10 μg/mlxanthorrizol, respectively.

As shown in FIGS. 4a, 4 b, 5 a and 5 b, it was clearly demonstrated thatthe efflux of protein, neucleic acid and ion was increased dramaticallyin 5 minutes after the treatment of xanthorrizol, and more efflux wasdetected in a group treated with 10 μg/ml xanthorrixol than a grouptreated with 5 μg/ml xanthorrizol. Given that the efflux ofintracellular macromolecules and ions was detected in the presence of 5μg/ml xanthorrizol, it could be appreciated that the overallexperimental data were consistent in a sense that antibacterial activityagainst Streptococcus mutans was also observed under the treatment of 5μg/ml xanthorrizol.

Example 3-4

Change in Cell Morphology After Xanthorrizol Treatment

To assess the extent of xanthorrizol-mediated cell damage, 10 μg/mlxanthorrizol was treated to the pathogens for 30 minutes. Then, cellmorphology was observed with transmission electron microscope (TEM). Incells treated with xanthorrizol, cell wall expansion was detected,followed by cell disruption accompanying the efflux of intracelluarmacromolecules from the cells. These results suggested thatxanthorrizol-mediated cell wall or cell membrane damage caused thedisruption in osmotic balance and physiological conditions, leading tocell death. Further, it was assumed that through the efflux ofintracellular macromolecules and ions, the permeability of cell wall isincreased to accelerate the efflux of intracellular components, which inturn inactivates cellular enzymes and causes the malfunction of cellwall.

EXAMPLE 4 Preparation of Tooth Paste Containing Xanthorrizol

Tooth paste containing xanthorrizol was prepared as following: first,alkyl sodium sulfate, sucrose fatty acid ester, sodiumcarboxymethylcellulose and. sodium chloride were dispersed in glycerinhomogeneously. Then, distilled water was added to dilute the mixture,followed by the addition of sodium. Then, xanthorrizol was added to themixture in a final concentration of 1.0, 0.1, 0.01 and 0.001%,respectively, and spice was further added under a vacuum condition togive cream-type tooth pastes, whose ingredients are disclosed in Table14.

TABLE 14 Tooth pastes containing xanthorrizol Tooth Tooth Tooth ToothIngredient Paste I Paste II Paste III Paste IV Xanthorrizol 1.0% 0.1%0.01%  0.001%  Sorbitol Solution 19.2%  19.2%  19.2%  19.2%  Glycerin22.0%  22.0%  22.0%  22.0%  Sucrose Fatty Acid Ester 2.0% 2.0% 2.0% 2.0%Alkyl Sodium Sulfate 1.7% 1.7% 1.7% 1.7% Sodium Chloride 1.0% 1.0% 1.0%1.0% Spice 1.1% 1.1% 1.1% 1.1% Sodium 0.4% 0.4% 0.4% 0.4%Carboxymethylcellulose Distilled Water q.s. q.s. q.s. q.s.

4 ml of each tooth paste containing xanthorrizol was taken totest-tubes, and mixed with 1 ml of dental pathogen cultures. Thetest-tubes were incubated with shaking, and antibacterial activity wasmeasured by counting viable cells with the lapse of time. Similarly,antibacterial activity of xanthorrizol-free tooth paste as a control wasalso determined against the dental pathogens. The results is are shownin Table 15.

TABLE 15 Antibacterial activity of tooth pastes containing xanthorrizolViable Cell Count (cell number/ml) Dental Pathogen Test Solution 0 hr 24hr Streptococcus Tooth Paste I 2.0 × 10⁵ <1 mutans Tooth Paste II 2.0 ×10⁵ <1 Tooth Paste III 2.0 × 10⁵ <1 Tooth Paste IV 2.0 × 10⁵ <1 Control2.0 × 10⁵ 3.1 × 10² Streptococcus Tooth Paste I 9.1 × 10⁵ <1 sobrinusTooth Paste II 9.1 × 10⁵ <1 Tooth Paste III 9.1 × 10⁵ <1 Tooth Paste IV9.1 × 10⁵ <1 Control 9.1 × 10⁵ 3.1 × 10² Porphyromonas Tooth Paste I 5.5× 10⁵ <1 gingivalis Tooth Paste II 5.5 × 10⁵ <5 Tooth Paste III 5.5 ×10⁵ 3.1 × 10² Tooth Paste IV 5.5 × 10⁵ 2.1 × 10³ Control 5.5 × 10⁵ 3.1 ×10⁴ Actinomyces Tooth Paste I 8.2 × 10⁵ <1 viscosus Tooth Paste II 8.2 ×10⁵ <1 Tooth Paste III 8.2 × 10⁵ <10  Tooth Paste IV 8.2 × 10⁵ 2.0 × 10²Control 8.2 × 10⁵ 3.1 × 10⁴

As clearly shown in Table 15, the tooth paste containing xanthorrizolexhibited higher antibacterial activity against the dental pathogens,compared to that of control which does not contain xanthorrizol.

EXAMPLE 5 Preparation of Oral Cleansers Containing Xanthorrizol

Several kinds of oral cleansers were prepared by the conventional methodto contain xanthorrizol in a concentration of 0.1%, 0.01%, 0.001% and0.0001%, respectively, whose components and composition ratios aredisclosed in Table 16.

TABLE 16 Oral cleansers containing xanthorrizol Oral Oral Oral Oral OralCleanser Cleanser I Cleanser II Cleanser III Cleanser IV Xanthorrizol0.1% 0.01% 0.001%  0.0001%  Sodium Fluoride 0.02%  0.02%  0.02%  0.02% Ethanol 6.6% 6.6% 6.6% 6.6% Glycerin 6.0% 6.0% 6.0% 6.0% /-menthol0.005%  0.005%  0.005%  0.005%  Sodium Saccharin q.s. q.s. q.s. q.s.Citric Acid q.s. q.s. q.s. q.s. Pigment q.s. q.s. q.s. q.s. Peppermintq.s. q.s. q.s. q.s. Essence Spearmint q.s. q.s. q.s. q.s. Essence

4 ml of each oral cleanser containing xanthorrizol with differentconcentrations was taken to test-tubes, and mixed with 1 ml of dentalpathogen cultures. The test-tubes were incubated with shaking, andantibacterial activity was measured with the lapse of time by countingviable cell numbers, whose results are shown in Table 17.

TABLE 17 Antibacterial activity of oral cleansers containingxanthorrizol Viable Cell Count (cell number/ml) Dental Pathogen TestSolution 0 hr 24 hr Streptococcus Oral cleanser I 2.0 × 10⁵ <1 mutansOral cleanser II 2.0 × 10⁵ <1 Oral cleanser III 2.0 × 10⁵ <1 Oralcleanser IV 2.0 × 10⁵ 3.2 × 10² Control 2.0 × 10⁵ 2.5 × 10⁴Streptococcus Oral cleanser I 9.1 × 10⁵ <1 sobrinus Oral cleanser II 9.1× 10⁵ <1 Oral cleanser III 9.1 × 10⁵ <1 Oral cleanser IV 9.1 × 10⁵ 1.1 ×10² Control 9.1 × 10⁵ 2.5 × 10⁴ Porphyromonas Oral cleanser I 5.5 × 10⁵<5 gingivalis Oral cleanser II 5.5 × 10⁵ <10  Oral cleanser III 5.5 ×10⁵ 1.7 × 10² Oral cleanser IV 5.5 × 10⁵ 5.5 × 10³ Control 5.5 × 10⁵ 2.5× 10⁴ Actinomyces Oral cleanser I 8.2 × 10⁵ <1 viscosus Oral cleanser II8.2 × 10⁵ <1 Oral cleanser III 8.2 × 10⁵ 1.2 × 10² Oral cleanser IV 8.2× 10⁵ 1.1 × 10³ Control 8.2 × 10⁵ 2.5 × 10⁴

As shown in Table 17, oral cleansers containing xanthorrizol were provento have higher antibacterial activity on dental pathogens causing cariesthan control group.

EXAMPLE 6 Preparation of Chewing Gum Containing Xanthorrizol

Gum base, starch hydrolysate, glucose, glycerin and sugar powder weremixed in a prewarmed blender. Then, to the mixture was addedxanthorrizol in a final concentration of 0.1, 0.01, 0.001 and 0.0001%,respectively, and mixed with spice and distilled water in the blenderwhose inner temperature was maintained at 55° C. for 20 minutes, finallyto give chewing gum. The ingredients of the chewing gum thus preparedare shown in Table 18.

TABLE 18 Chewing gum containing xanthorrizol Chewing Chewing ChewingChewing Chewing Gum Gum I Gum II Gum III Gum IV Xanthorrizol 0.1% 0.01%   0.001%   0.0001%    Gum Base 20% 20% 20% 20% Sugar Powder 54%54% 54% 54% Starch 10% 10% 10% 10% Hydrolysate Glycerin  5%  5%  5%  5%Spice q.s. q.s. q.s. q.s. Distilled Water q.s. q.s. q.s. q.s.

EXAMPLE 7 Preparation of Antibacterial Soap Containing Xanthorrizol

Xanthorrizol was mixed with soap base to a final concentration of0.001%, 0.01%, 0.1%, 1.0% and 5.0%(w/w), respectively. In accordancewith the conventional method, antibacterial soap was prepared by addingspice and pigment to the mixture. Table 19 discloses the ingredients ofantibacterial soap.

TABLE 19 Antibacterial soap containing xanthorrizol Antibacterial SoapSoap I Soap II Soap III Soap IV Soap V Xanthorrizol 0.001% 0.01% 0.1%1.0% 5.0% Soap Base q.s. q.s. q.s. q.s. q.s. Spice q.s. q.s. q.s. q.s.q.s. Pigment q.s. q.s. q.s. q.s. q.s.

Each antibacterial soap containing xanthorrizol with differentconcentrations was taken to 4 ml of medium, and mixed with 1 ml of skininfectious pathogen cultures. The test-tubes were incubated withshaking, and antibacterial activity was measured after 48 hour bycounting viable cells. As a control, antibacterial activity ofxanthorrizol-free soap was also examined in an analogous manner. Theresults are disclosed in Table 20.

TABLE 20 Antibacterial activity of soap containing xanthorrizolAcne-causing Test Viable Cell Count (cell number/ml) Pathogen Solution 0hr 24 hr Propionibacterium Soap I 2.0 × 10⁵ 1.2 × 10⁴ acnes Soap II 2.0× 10⁵ <10  Soap III 2.0 × 10⁵ <1 Soap IV 2.0 × 10⁵ <1 Soap V 2.0 × 10⁵<1 Control 2.0 × 10⁵ 4.3 × 10³ Candida albicans Soap I 4.4 × 10⁵ 5.1 ×10³ Soap II 4.4 × 10⁵ <20  Soap III 4.4 × 10⁵ <1 Soap IV 4.4 × 10⁵ <1Soap V 4.4 × 10⁵ <1 Control 4.4 × 10⁵ 4.1 × 10² Staphylococcus Soap I4.2 × 10⁵ 2.3 × 10⁴ aureus Soap II 4.2 × 10⁵ <10  Soap III 4.2 × 10⁵ <1Soap IV 4.2 × 10⁵ <1 Soap V 4.2 × 10⁵ <1 Control 4.2 × 10⁵ 4.5 × 10⁵Staphylococcus Soap I 5.8 × 10⁵ 1.2 × 10⁴ epidermis Soap II 5.8 × 10⁵<15  Soap III 5.8 × 10⁵ <1 Soap IV 5.8 × 10⁵ <1 Soap V 5.8 × 10⁵ <1Control 5.8 × 10⁵ 6.2 × 10⁵

As shown in Table 20, soap containing xanthorrizol exerted strongerantibacterial effects on skin infectious pathogens than control.

EXAMPLE 8 Preparation of Facial Lotion Containing Xanthorrizol

To prepare facial lotion of the invention, xanthorrizol was first mixedwith water in 4 different concentrations of 1.0, 0.1, 0.01 and 0.001%,respectively, and subsequently with phosphate solution. Then, ethanol,glycerin and propyleneglycol were added to the said mixtures togetherwith spices and preservatives. And then, to the mixtures was added waterto give homogeneous mixtures with equal amounts, whose particularingredients are disclosed in Table 21.

TABLE 21 Facial lotion containing xanthorrizol Facial Lotion Lotion ILotion II Lotion III Lotion IV Xanthorrizol 1.0% 0.1% 0.01%  0.001% Glycerin 2.0% 2.0% 2.0% 2.0% Propyleneglycol 2.0% 2.0% 2.0% 2.0% CalciumPhosphate 1.0% 1.0% 1.0% 1.0% Disodium Phosphate 0.05%  0.05%  0.05% 0.05%  Spice 0.02%  0.02%  0.02%  0.02%  96% Ethanol  50%  50%  50%  50%Distilled Water q.s. q.s. q.s. q.s. Preservative q.s. q.s. q.s. q.s.

4 ml of each facial lotion containing xanthorrizol was taken totest-tubes, and mixed with 1 ml of acne-causing pathogen cultures. Thetest-tubes were incubated with shaking, and antibacterial activity wasmeasured after 24 hours by counting viable cell numbers, whose resultsare shown in Table 22.

TABLE 22 Antibacterial activity of facial lotion containing xanthorrizolAcne-causing Viable Cell Count (cell number/ml) Pathogen Test Solution 0hr 24 hr Propionibacterium Lotion I 3.0 × 10⁵ <1 acnes Lotion II 3.0 ×10⁵ <1 Lotion III 3.0 × 10⁵ <1 Lotion IV 3.0 × 10⁵ 1.2 × 10² Control 3.0× 10⁵ 3.1 × 10³ Staphylococcus Lotion I 4.5 × 10⁵ <1 aureus Lotion II4.5 × 10⁵ <1 Lotion III 4.5 × 10⁵ <1 Lotion IV 4.5 × 10⁵ 3.1 × 10²Control 4.5 × 10⁵ 4.7 × 10³ Staphylococcus Lotion I 6.8 × 10⁵ <1epidermis Lotion II 6.8 × 10⁵ <1 Lotion III 6.8 × 10⁵ <1 Lotion IV 6.8 ×10⁵ 3.5 × 10² Control 6.8 × 10⁵ 5.1 × 10²

As clearly demonstrated in Table 22, facial lotion containingxanthorrizol was proven to have higher antibacterial activity againstacne-causing pathogens, compared to that of control groups.

EXAMPLE 9 Preparation of Facial Cream Containing Xanthorrizol

Facial creams containing xanthorrizol were prepared in accordance withthe components and composition ratios shown in Table 23: first,substances B and C were each melted down at the temperature of 75 to 80°C., followed by emulsification of the melted substance C in the meltedsubstance B. Then, xanthorrizol was added to the mixtures in aconcentration of 3.0, 0.1, 0.01 and 0.001%, respectively. Finally, spicewas added to the mixtures which were adjusted to the final volume withdistilled water.

TABLE 23 Facial cream containing xanthorrizol Facial Cream Cream I CreamII Cream III Cream IV A Xanthorrizol 3.0% 0.1% 0.01%  0.001%  B Glycerin2.0% 2.0% 2.0% 2.0% Propyleneglycol 2.0% 2.0% 2.0% 2.0% Chlorolauryl8.0% 8.0% 8.0% 8.0% Sulfate Stearin 5.4% 5.4% 5.4% 5.4% Mineral Oil 4.5%4.5% 4.5% 4.5% C Spice 0.02%  0.02%  0.02%  0.02%  Cetyl Alchohol q.s.q.s. q.s. q.s. Distilled Water q.s. q.s. q.s. q.s. Preservative q.s.q.s. q.s. q.s.

4 ml of each cream containing the crude extract was taken to test-tubes,and mixed with 1 ml of acne-causing pathogen cultures. The test-tubeswere incubated with shaking, and antibacterial activity in test-tubeswas measured after 24 hours by counting viable cell numbers. The resultsare shown in Table 24.

TABLE 24 Antibacterial activity of facial creams containing xanthorrizolAcne-causing Viable Cell Count (cell number/ml) Pathogen Test Solution 0hr 24 hr Propionibacterium Cream I 3.0 × 10⁵ <1 acnes Cream II 3.0 × 10⁵<1 Cream III 3.0 × 10⁵ <1 Crean IV 3.0 × 10⁵ 1.5 × 10² Control 3.0 × 10⁵3.3 × 10⁴ Staphylococcus Cream I 4.5 × 10⁵ <1 aureus Cream II 4.5 × 10⁵<1 Cream III 4.5 × 10⁵ <1 Cream IV 4.5 × 10⁵ 3.4 × 10⁴ Control 4.5 × 10⁵4.1 × 10³ Staphylococcus Cream I 6.8 × 10⁵ <1 epidermis Cream II 6.8 ×10⁵ <1 Cream III 6.8 × 10⁵ <1 Cream IV 6.8 × 10⁵ 4.3 × 10² Control 6.8 ×10⁵ 5.1 × 10³

As clearly demonstrated in Table 24, facial creams exhibited muchstronger antibacterial activity against acne-causing pathogens, comparedto that of the control groups.

EXAMPLE 10 Skin Safety Test

To test safety of the crude extract and xanthorrizol havingantibacterial activity to human skin, a patch test was performed byemploying 0.3% crude extract, 0.06% crude extract, 0.06% xanthorrizol,and petrolatum as a control: after 6 healthy volunteers wore patches ontheir skin for 48 hours, skin conditions were observed for 48 to 96hours in terms of adverse effects caused by the patches(see: Agner T.,Clinical grading of experimental skin reactions. In “Handbook ofnon-invasive methods and the skin”, Serup, J.(ed.), CRC press, 575-578,1995), whose results are summarized in Table 25.

TABLE 25 Patch test for crude extract and xanthorrizol ResponseConcentration False Positive Weak Positive Substance (%) ResponseResponse Crude extract 0.3% — — Crude extract 0.06% — — Xanthorrizol0.06% — — Control (vaseline) — —

As shown in Table 25, it was clearly demonstrated that both ofxanthorrizol and crude extract do not cause adverse effects such aserythema, scarring or edema on skin, assuring their safe use for variousantibacterial products.

As clearly illustrated and demonstrated as above, the present inventionprovides a process for producing xanthorrizol from Curcuma xhanthorrizaRoxb. and novel uses of the same. Xanthorrizol of the present inventionhas a strong antibacterial activity with a broad spectrum under a hightemperature. Therefore, xanthorrizol can be applied as an activeingredient for various antibacterial products such as antibacterialagent, tooth paste, oral cleanser, chewing gum, soap and cosmetics.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purpose, those who are skilled in the artwill appreciate that various modifications, additions and substitutionsare possible, without departing from the scope and spirit of theinvention as disclosed in the accompanying claims.

What is claimed is:
 1. An antibacterial composition comprising anantibacterially effective amount of xanthorrizol.
 2. The antibacterialcomposition of claim 1, wherein the composition is formulated in apharmaceutical dosage form selected from the group consisting of tablet,capsule, soft gelatin capsule, solution, ointment, plaster, granule,lotion, aerosol, suppository, cataplasma, cream, troche, paste, pill andinjection.
 3. The antibacterial composition of claim 1, wherein theantibacterial composition is formulated in an oral product.
 4. Theantibacterial composition of claim 3, wherein the oral product comprisesa toothpaste, an oral cleanser or a chewing gum.
 5. The antibacterialcomposition of claim 4, wherein the toothpaste comprises 0.001 weight %to 1.0 weight % of the xanthorrizol based on the total weight of thetoothpaste.
 6. The antibacterial composition of claim 4, wherein theoral cleanser comprises 0.0001 weight % to 0.1 weight % of thexanthorrizol based on the total weight of the oral cleanser.
 7. Theantibacterial composition of claim 4, wherein the chewing gum comprises0.0001 weight % to 0.1 weight % of the xanthorrizol based on the totalweight of the chewing gum.
 8. The antibacterial composition of claim 1,wherein the antibacterial composition is formulated in a product of soapor cosmetics.
 9. The antibacterial composition of claim 8, wherein thesoap comprises 0.001 weight % to 5.0 weight % of the xanthorrizol basedon the total weight of the soap.
 10. The antibacterial composition ofclaim 8, wherein the cosmetics comprises 0.001 weight % to 3.0 weight %of the xanthorrizol based on the total weight of the cosmetics.
 11. Theantibacterial composition of claim 1, wherein the xanthorrizol isextracted from Curcuma Xanthorriza Roxb.
 12. The antibacterialcomposition of claim 1, wherein the xanthorrizol is included in an crudeextract of Curcuma Xanthorriza Roxb.
 13. The antibacterial compositionof claim 12, wherein the crude extract is obtained by an extraction, theextraction being selected from the group consisting of an organicsolvent extraction, a supercritical fluid extraction, a microwaveextraction and an ultrasonic extraction.
 14. The antibacterialcomposition of claim 13, wherein the organic solvent extraction iscarried out by using an organic solvent, the organic solvent beingselected from the group consisting of methanol, ethanol, hexane,ethylacetate, chloroform and mixtures thereof.
 15. The antibacterialcomposition of claim 13, wherein the supercritical fluid extraction iscarried out by using carbon dioxide as a supercritical fluid under apressure of 100 to 400 bar, at a temperature of 35 to 70° C.
 16. Theantibacterial composition of claim 13, wherein the microwave extractionis carried under a microwave condition of 2,450 MHz for 1 to 10 minutesat a temperature of 50 to 120° C., using a solvent, the solvent beingselected from the group consisting of water, ethanol, methanol andhexane.
 17. The antibacterial composition of claim 13, wherein theultrasonic extraction is carried under a microwave condition of 2,450MHz. for 1 to 10 minutes at a temperature of 50 to 120° C., using asolvent, the solvent being selected from the group consisting of water,ethanol, methanol and hexane.